Understanding how membrane lipids influence membrane organization and function remains one of the most important challenges in membrane biology. Artificial vesicles composed of membrane lipids have been widely used to study membranes, but generally lack lipid asymmetry, the difference in lipid composition in the inner and outer membrane leaflets (monolayers) characteristic of most biomembranes. Our lab developed a breakthrough method allowing preparation of a wide variety of lipid vesicles with highly controllable lipid asymmetry. Asymmetric vesicles will be used to define principles of membrane organization and function that are of biomedical relevance. Using microscopy and fluorescence spectroscopy the effect of lipid composition and asymmetry upon the organization of membrane lipids and proteins into domains with different molecular compositions will be defined. In 1994 we proposed, together with Dr. Deborah Brown (Stony Brook), the widely used working model for the nature of eukaryotic cell domains: that they are sphingolipid- and sterol-rich liquid ordered-like domains co-existing with disordered domains rich in unsaturated lipids. Since sphingolipids are only abundant in outer leaflets, domain formation in inner leaflets is likely to involve interactions between the leaflets, i.e. coupling between inner and outer leaflet physical properties. Preliminary studies confirm that this interleaflet coupling occurs. First, the hypothesis that in asymmetric membranes that mimic plasma membranes lipid domains in the outer leaflet induce spontaneous formation of inner leaflet lipid domains will be tested. Then the effect of lipid structure upon the domain formation and interleaflet coupling will be defined. Lipi acyl chain length, unsaturation, sterol concentration, and sterol structure will be varied. This should define what cell membranes have lipids with the capacity to spontaneously form domains in the outer and/or inner leaflets. These studies will have biomedical implications. Testing the hypothesis that lipids with one very long acyl chain, which allows penetration from one leaflet into another (i.e. interdigitation), enhance coupling of physical properties will have implications for adrenoleukoadenopathy, a disease which causes lipids to massively overaccumulate interdigitating acyl chains. In addition, the studies will test the hypothesis that membrane organization is impacted in diseases involving cholesterol biosynthesis defects resulting in massive overaccumulation of precursor sterols, e.g. Smith-Lemli-Opitz syndrome and desmosterolosis. The next studies will examine how membrane protein sequence controls domain formation and the association of membrane proteins with specific domains. Finally, lipids which we find modulate domain formation in the studies above and which can be exchanged into cells will be used as tools to probe the functional consequences of domain formation and interleaflet coupling in cells. The hypothesis that endocytosis and the clustering of inner leaflet lipids involved in signal transduction requires interleaflet coupling linked to outer leaflet domain formation will be tested.